Foodborne diarrheal disease cause 76,000,000 cases each year in the United States. Intestinal pathology caused by Shiga toxin-producing Escherichia coli (EHEC), important food-borne pathogens, includes approximately 35% of all bloody diarrhea in the United States, an unknown % of watery diarrhea, and the life-threatening systemic manifestations (observed in up to 10% of cases) of infection, the hemolytic uremic syndrome (HUS) and encephalopathy. EHEC are the leading cause currently of acute and chronic renal failure in children in the USA. EHEC is a particularly worrisome foodborne pathogen, because the number of outbreaks caused by EHEC continue to significantly increase and there is no effective specific therapy for this illness which is lethal in up to 10% of children who develop HUS. Thus, there is a great need to better understand the pathogenesis of this infection to promote development of new therapeutic approaches to treat EHEC infection and its complications. Understanding the complex mechanism of Stx uptake and trafficking pathways into colonic epithelium may help to identify new drug targets and develop new strategies directed at preventing toxin action on human intestine. In this application we will study the mechanism of Stx1 and its B-subunit (Stx1B) uptake, trafficking and intracellular action using animal and human intestinal epithelial cell models. In Aim 1 we will study the role of lipid rafts (LR) in Stx1/Stx1B translocation across the apical cell surface. We propose to test the role of LR proteins, which are associated with toxin receptor in toxin translocation machinery. In Aim 2 we will study a newly recognized Stx1/Stx1B internalization pathway from plasma membrane into the nucleoli. In Aim 3 we will study the mechanisms of Stx1/Stx1B-induced apoptosis and its role in toxin-related intestinal inflammation. These proposed studies should provide new insight into the understanding of the molecular basis of Stx-mediated intestinal epithelial cell injury and facilitate the design of strategies to prevent Stx1 uptake and intracellular action, and thus serve to find new targets to interfere with EHEC-related gastrointestinal pathophysiology.